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Abstract Aminoglycosides (AGs) are broad-spectrum antibiotics effective against Gram-negative bacteria, with uptake dependent on membrane potential. However, the mechanisms of AG entry remain incompletely understood. Here, we identify a previously undescribed uptake pathway via carbohydrate transporters in E. coli. By deleting or overexpressing 26 carbohydrate transporters, we found that 18 facilitated AG uptake, a mechanism conserved across several Gram-negative ESKAPEE pathogens.
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